The primary role of salivary glands is to produce saliva, which provides much of the innate host defense in the oral cavity. Clinically, salivary hypofunction causes significant morbidity and there is no conventional treatment for irreversible gland loss. The purpose of this study is to identify and isolate stem cells from adult human salivary glands. Human submandibular glands were treated enzymatically to produce single cell suspensions and plated at clonal densities on Type IV collagen-coated tissue culture plastic to encourage attachment of epithelial cells, and incubated in low calcium (0.1 mM) serum-free nutrient medium. Clonal cells expanded in low calcium medium were characterized by immunocytochemistry, and subsequently were placed into wells containing 50% Matrigel, normal calcium (1.2 mM), and FGF-7 or FGF-10. Cells were also transplanted into immunocompromised mice with a polyglycolic acid scaffolding with Matrigel as a carrier, harvested after two months, and characterized by immunohistochemistry. [unreadable] [unreadable] Several clones were isolated. In low calcium, the cells proliferated rapidly (until passage 10), and displayed a mesenchymal rather than epithelial morphology. Cells that were derived from a single clone expressed both pan-cytokeratin (an epithelial marker) and vimentin (a mesenchymal marker). When grown in low calcium medium, cells did not express acinar cell-specific mRNAs, but in normal calcium conditions, they expressed both ductal and acinar mRNA markers. In Matrigel, these cells aggregated and FGF-10 induced branching and globular outgrowth from the aggregates, as well as expression of ductal cell-specific protein. Transplanted cells made both ductal- and acinar-like structures, and expressed both ductal and acinar markers (both mRNAs and proteins). Cells obtained from enzymatically dispersed human submandibular glands appear to include progenitors, a subset of which may be able to form both ductal and acinar phenotypes. [unreadable] [unreadable] To label a putative stem cell (slowly-dividing, label retaining cell) in vivo, BrdU was injected into young-adult mice intraperitoneally for 18 days. This extended series of injections is needed to increase the chance that the stem cell, which rarely divides, to be labeled. On the last day of injection, and then at 0.5, 1, 2, 4, 6, and 7 months later, submandibular salivary glands were harvested in order to watch the disappearance of labeled cells (differentiated cells and transiently dividing progenitor cells) and to identify the location of the label-retaining cell, which would be a putative salivary stem cell. From 7 days to 100 days, label-retaining cells range from 70% to 30% of the total number of cells respectively. But after 152 days, the label-retaining cells are less than 3% of the total cells. Label-retaining cells are not only ductal cells but also acinar cells, and a few label-retaining cells are non-ductal, non-acinar, and non-myoepithelial in nature. After 186 days, the label-retaining cells have disappeared from the salivary gland. These results suggest that a subset of acinar cells, striated and granular duct cells, and a cell type of unknown identity are slowly dividing, label-retaining cells that serve as reserve cell pools, perhaps by self-renewal. After injury, it is thought that a stem cell will divide immediately in order to initiate regeneration. In future studies, we will aim to characterize this cell type by immunohistochemical staining to identify a set of markers for this cell type. [unreadable] [unreadable] Currently, we are determining the expression of ductal and acinar markers of the cell aggregates and in vivo transplants by RT-PCR and immunohistochemistry. Again, we are paying particular attention to the expression of cell surface markers that could be used for the cell sorting experiments. There are still some critical experiments that must be performed before this study is complete, and that is to determine if the cells are able to actually function. In order to do this, two experiments have been planned. The first is to determine if the cells will incorporate into an injured salivary gland and form appropriate connections with the residual gland. The second is to destroy saliva-producing acinar cells in mice by irradiation (this is a major problem in patients undergoing radiation for the treatment of head and neck cancer), thereby inducing dry mouth. The cells will then be transplanted around the residual ductal cells (which do not produce saliva), and determine if gland can be reformed by transplanted cells, and if saliva is produced.